Skeletal muscle insulin resistance plays a primary role in the development of type 2 diabetes and may be causally associated with inflammation and altered lipid metabolism. Circulating levels of fatty acids and pro-inflammatory cytokines are elevated in obese, diabetic subjects and shown to cause insulin resistance in skeletal muscle. In contrast, plasma levels of IL-10, an anti-inflammatory cytokine, are positively related to insulin sensitivity and reduced in subjects with metabolic syndrome. We have recently shown that acute treatment with IL-10 prevents lipid-mediated insulin resistance in muscle that is associated with increased insulin signaling in mice. To further examine the role of IL-10 in skeletal muscle insulin action, we have recently generated transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10 mice). Our preliminary data indicate that MCK-IL10 mice are protected from lipid- mediated defects in insulin signaling and glucose metabolism in muscle. We also find that diet-induced insulin resistance is associated with increased macrophage infiltration in skeletal muscle, and these effects are attenuated in MCK-IL10 mice. We hypothesize that IL-10 prevents muscle insulin resistance by 1) blocking lipid-mediated activation of PKC-8/JNK/IKK and down regulation of insulin signaling, and/or 2) suppressing obesity-associated macrophage infiltration and inhibiting the deleterious effects of macrophage-derived cytokines on glucose metabolism. Based on our preliminary data showing altered intramuscular lipid levels in IL-10 treated mice, the Aim 1 will examine the effects of muscle IL-10 overexpression on glucose and lipid metabolism. In Aim 2, we will identify the mechanism by which MCK-IL10 mice are protected from lipid-mediated insulin resistance using chronic high-fat feeding, acute lipid infusion, and genetically obese mouse models. The Aim 3 will determine the role of macrophage infiltration in muscle insulin resistance in diet-induced obesity and acute lipid infusion models. We will also observe macrophage migration using monocyte labeling and examine the effects of macrophage depletion using clodronate on lipid-mediated insulin resistance. Lastly, we will investigate the role of alternatively-activated macrophages in MCK-IL10 phenotypes. Overall, our proposed studies will identify a novel role of IL-10 in the regulation of skeletal muscle insulin action and discover new therapeutic targets in the treatment of insulin resistance and type 2 diabetes. PUBLIC HEALTH RELEVANCE. Skeletal muscle insulin resistance plays a major role in the development of type 2 diabetes, which impacts more than 170 million people worldwide and may be causally associated with inflammation and altered lipid metabolism. The proposed studies will examine the mechanisms by which interleukin-10 regulates skeletal muscle insulin action and prevents lipid-mediated insulin resistance in skeletal muscle. Our findings will provide important insights into the role of obesity and inflammation in insulin resistance and further identify novel therapeutic targets in the treatment of type 2 diabetes.